The CGG repeat expansion in the 5′-UTR of the fragile X mental retardation gene (FMR1) has been implicated in the pathogenesis of two distinct disorders, fragile X syndrome (FXS), a neurodevelopmental disorder and fragile X-associated tremor and ataxia syndrome (FXTAS), a progressive neurodegenerative disease that is usually late onset. While normal individuals generally possess between 5 and 54 CGG repeats, fully affected individuals have more than 200 CGG repeats on what are referred to as “full mutation alleles.” “Pre-mutation alleles” (55-200 CGG repeats) of the FMR1 gene are known to contribute to the fragile X phenotype through genetic instability and could expand into full mutation during germline transmission.
FXTAS has been recognized among many male pre-mutation carriers in or beyond their fifth decade of life and is uncoupled from the FXS neurodevelopmental disorder. Although both disorders involve repeat expansions in the FMR1 gene, the clinical presentation and molecular mechanisms underlying each disease are distinct. The most common clinical feature of FXTAS is a progressive action tremor with ataxia. More advanced or severe cases may show a progressive cognitive decline that ranges from executive and memory deficits to dementia. Patients may also present with common psychiatric symptoms such as increased anxiety, mood liability and depression. Patients also complain of fluctuating muscle weakness and numbness and/or pain in the lower extremities, which suggests the disease may not be purely neurological.
Magnetic resonance imaging (MRI) of adult male patients affected with FXTAS demonstrated mild to moderate global brain atrophy, most common in the fontal and parietal regions as well as the pons and cerebellum. The most significant radiological findings were the increased T2 intensities of the middle cerebellar peduncle (MCP) and adjacent cerebellar white matter not seen in controls.
Nearly all case studies on autopsy brains of symptomatic premutation carriers demonstrated degeneration in the cerebellum, including Purkinje neuronal cell loss, Bergman gliosis, spongiosis of the deep cerebellar white matter and swollen axons. The major neuropathological hallmark and postmortem criterion for definitive FXTAS is eosinophilic, ubiquitin-positive intranuclear inclusions located in broad distribution throughout the brain in neurons, astrocytes, and in the spinal column. The inclusions are both tau and α-synuclein negative, which indicates that FXTAS is not a tauopathy or synucleinopathy. The FXTAS inclusions share the ubiquitin positive hallmark with several other inclusion disorders, such as polyglutamine disorders, although the inclusions do not stain with antibodies that recognize polyglutamine, which suggests a defect in the proteasomal degradation pathway. Unlike the polyglutamine disorders, there is no known structurally abnormal protein produced in FXTAS (the premutation is non-coding).
An RNA gain-of-function mechanism has been suggested for FXTAS based on the observation of increased levels of CGG-containing FMR1 mRNA, along with either reduced FMRP in premutation carriers. The absence of FXS, which results from the loss of function of the FMR1 gene product, in FXTAS patients along with absence of symptoms in older individuals with FXS also suggests a role for the expanded ribo-CGG (rCGG) repeat in FXTAS pathology. This type of RNA gain of function mechanism has been suggested as a mechanism for triplet repeat-related ataxias such as SCA8, SCA10, and SCA12 and in myotonic dystrophy (DM). The untranslated repeat expansion in DM has offered major insight into the underlying molecular mechanisms of FXTAS. DM1 is caused by a CTG repeat expansion in a region of transcribed RNA, but not translated into protein, the 3′UTR of the DMPK gene. The mutant transcripts sequester certain proteins, which form ribonuclear foci or inclusions.
Several additional lines of evidence further support an RNA-mediated gain-of-function toxicity model for FXTAS. First, in a “knock-in” mouse model designed with a ˜100 CGG repeat fragment, intranuclear inclusions were found to be present throughout the brain. An increase in both the number and size of the inclusions was observed during the life course, which correlates with the progressive character of the phenotype observed in humans. Neuropathological studies in humans have revealed a highly significant association between length of the CGG tract and frequency of intranuclear inclusions in both neurons and astrocytes, indicating that the CGG repeat length is a powerful predictor of neurological involvement and mortality. Intranuclear inclusions can be formed in both primary neural progenitor cells and established neural cell lines with premutation CGG repeat. A model of FXTAS using Drosophila has been described and it was demonstrated that premutation-length riboCGG (rCGG) repeats are both toxic and sufficient to cause neurodegeneration. These observations led to the proposal that transcription of the CGG90 repeats leads to an RNA-mediated neurodegenerative disease and that rCGG repeat-binding proteins (RBPs) become functionally limited by their sequestration to lengthy rCGG repeats, mechanistically similar to the pathophysiology of DM1.
Drosophila has emerged as a premiere model system for the study of human neurodegenerative diseases within the last decade due to the realization that flies and humans share many structurally and functionally related gene families. It has been shown that genes associated with neurodegeneration could be expressed in flies, causing phenotypes remarkably similar to those of the counterpart human diseases, including poly-glutamine disorders, Parkinson's disease and Alzheimer's disease. These results indicate that the molecular mechanisms of neuronal toxicity and loss are conserved between human and flies. Development of such disease models in the fly allows genetic approaches to be applied to address specific hypotheses concerning disease progression and to test candidate modifier genes or therapeutic drug compounds.
Given the high prevalence of fragile X premutation carriers among the general population (˜1 in every 800 males and 250 females) and the high risk of developing FXTAS among the male carriers, it is important to develop new therapeutic interventions for FXTAS.